Process for aligning macromolecules by passage of a meniscus and applications

ABSTRACT

The subject of the present invention is a process for aligning a macromolecule (macromolecules) on the surface S of a support, characterized in that the triple line S/A/B (meniscus) resulting from the contact between a solvent A and the surface S and a medium B is caused to move on the said surface S, the said macromolecules having a part, especially an end, anchored on the surface S, the other part, especially the other end, being in solution in the solvent A.  
     The subject of the present invention is also a process for detecting, measuring the intramolecular distance of, separating and/or assaying a macromolecule in a sample in which a process of alignment according to the invention is used.

[0001] The present invention relates to a method for aligningmacromolecules such as polymers or macromolecules with biologicalactivity, especially DNA, or proteins. The present invention alsorelates to the application of this method in processes for detecting formeasuring intramolecular distance, for separating and/or for assaying amacromolecule in a sample.

[0002] Controlling the conformation of macromolecules represents a majorindustrial challenge, for example in the manufacture of sensors or ofcontrolled molecular assemblies, or alternatively in problems ofdetection and analysis. It may be useful to have an elongated molecularconformation. By way of example, in the case where polymers are graftedon a substrate, it has been proposed to extend them by the action of anelectric field, a flow or with the aid of optical tweezers. Inparticular, in biology, the alignment of DNA—by electrophoresis(Zimmermann and Cox Nucl. Acid Res. 22, p 492, 1994), free flow (Parraand Windle, Nature Genetics, 5, p 17, 1993 and WO 93/22463) or in a gel(Schwartz et al. Science 262, p 110, 1993 and U.S. Pat. No. 33,531) orwith the aid of optical tweezers (Perkins et al., Science 264 p 819,1994 and also U.S. Pat. No. 5,079,169)—opens numerous possibilities inmapping, or in the detection of pathogens.

[0003] These methods only allow in general an imperfect alignment, oralternatively a transient alignment—that is to say that relaxation ofthe molecule occurs once the stress disappears. In the case of opticaltweezers, the method is expensive, is limited to only one molecule at atime, and is difficult to carry out by non-qualified staff.

[0004] A special technique for aligning DNA by flow after cell lysis,followed by drying, has been proposed (I. Parra and B. Windle and WO93/22463). The alignment obtained is very imperfect and nonhomogeneousand numerous nonaligned masses are observed.

[0005] The subject of the present invention is a novel and simple methodfor aligning macromolecules on the surface S of a support, characterizedin that the triple line S/A/B (meniscus) resulting from the contactbetween a solvent A and the surface S and a medium B is caused to moveon the said surface S, the said macromolecules having a part, especiallyan end, anchored on the surface S, the other part, especially the otherend, being in solution in the solvent A.

[0006] It has been observed according to the present invention that themere passage of a meniscus over molecules of which one part is anchoredon a substrate, the remainder of the molecule existing freely insolution makes it possible to align them uniformly, perpendicularly tothe moving meniscus, leaving them adsorbed on the surface behind themeniscus. This phenomenon is called “molecular combing” here.

[0007] More specifically, the stretching of the free part of themolecule is achieved by the passage of the triple line S/A/Bconstituting the meniscus between the surface S, the solvent A and amedium B which may be a gas (in general air) or another solvent.

[0008] In a specific embodiment, the meniscus is a water/air meniscus,that is to say that the solvent A is an aqueous solution and the mediumB is air.

[0009] Furthermore, it is possible to extend the air/water meniscus usedhere in order to stretch the molecule to other systems such as oil/wateror water/surfactant/air, in particular.

[0010] The movement of the meniscus can be achieved by any means ofrelative movement of the fluids A and B relative to the surface S. Inone embodiment, the surface S can be removed from the solvent A orconversely, the solvent A can be removed from the surface S.

[0011] In particular, the meniscus can be, moved by mechanical means,especially by pneumatic means by aspirating or blowing a gas, orespecially by hydraulic means by pushing or aspirating the solvent A orthe medium B.

[0012] Thus, the movement of the meniscus can be achieved by gradualevaporation of the solvent A.

[0013] When the movement of the meniscus is achieved mechanically, itcan be achieved either by translation of the interface A/B, or bytranslation of the surface S.

[0014] In a specific embodiment, the solvent is placed between twosupports of which at least one corresponds to the said support ofsurface S and the meniscus is moved for example by evaporation.

[0015] By “support”, there is understood here any substrate whosecohesion is sufficient to withstand the passage of the meniscus.

[0016] The support may consist, at least at the surface, of an organicor inorganic polymer, a metal especially gold, a metal oxide or sulfide,a semiconductor element or an oxide of a semiconductor element, such assilicon oxide or a combination thereof, such as glass or a ceramic.

[0017] There may be mentioned more particularly glass, surface oxidizedsilicon, graphite, mica and molybdenum sulfide.

[0018] As “support”, there may be used a single support such as a slide,beads, especially polymer beads, but also any form such as a bar, afiber or a structured support, and also particles, whether it bepowders, especially silica powders, which can moreover be made magnetic,fluorescent or colored as known in the various assay technologies.

[0019] The support is advantageously in the form of cover slips.Preferably, the support has little or no fluorescence.

[0020] Macromolecules, such as ordinary polymers, or biological polymerssuch as DNA, RNA or proteins, can be anchored by ordinary methods on asupport.

[0021] The macromolecule to be aligned can be chosen from biologicalmacromolecules such as proteins, especially antibodies, antigens,ligands or their receptors, nucleic acids, DNA, RNA or PNA, lipids,polysaccharides or derivatives thereof.

[0022] It was observed according to the present invention, that thestretching force acts locally within the immediate vicinity of themeniscus. It is independent of the length of the molecule, of the numberof molecules anchored, and within a wide range, of the speed of themeniscus. These characteristics are particularly important for aligningthe molecules homogeneously and reproducibly.

[0023] It is possible, according to the present invention, to addsurfactant elements into the solvent A and/or the medium B, which modifythe properties of the interfaces. According to the present invention,the stretching can indeed be controlled by the addition of surfactants,or by an adequate surface treatment.

[0024] Too high a surface-macromolecule attraction (for example anexcessively high level of adsorption) can interfere with the alignmentof the molecules by the meniscus, these molecules remaining adsorbed atthe surface in a state which is not necessarily stretched. Preferably,the surface exhibits a low rate of adsorption of the said macromolecule,such that only the anchored molecules will be aligned, the others beingcarried by the meniscus.

[0025] However, it is possible to vary the differences in adsorptionbetween a part of the macromolecule, especially its ends, and its otherparts (in particular for long molecules such as DNA or collagen) inorder to anchor, by adsorption, the molecules by a part, especiallytheir end(s) alone, the remainder of the molecule existing freely insolution, on a wide variety of surfaces and align them by the passage ofthe meniscus as described above.

[0026] The adsorption of a macromolecule onto a surface can be easilycontrolled by means of the pH or of the ionic content of the medium orof an electric voltage applied over the surface. The surface charges andthe electrostatic (repulsive or attractive) interactions between thesurface and the molecule are thus changed, thereby making it possible topass from a state of complete adsorption of the molecule onto thesurface to a total absence of adsorption. Between these two extremecases, there is a range of control parameters where the adsorptionoccurs preferably through the end of the molecules and which willtherefore be used advantageously to anchor them on the surface, and thento align them by the passage of the meniscus.

[0027] Once aligned, the molecules adhere strongly to the surface. Inthe case of DNA, it was possible to observe them by fluorescence severalmonths after their alignment.

[0028] The present invention is therefore very different from the methodproposed by Parra and Windle, because according to the presentinvention, the molecules are anchored on the surface and then uniformlyaligned by the passage of the meniscus, whereas in the Parra and Windlemethod, a hydrodynamic flow is used to stretch the moleculesnonhomogeneously, which molecules will become nonspecifically adsorbedonto the surface.

[0029] Other techniques can also result in the stretching and thealignment of molecules. Thus, a dynamic orientation of molecules insolution, anchored at one end, can be obtained by electrophoresis or bya hydraulic flow. However, the results observed show that thesetechniques are much less efficient than the use of the meniscus.

[0030] By “anchoring” of the macromolecule on the surface, there shouldbe understood an attachment resulting from a chemical reactivity boththrough a covalent linkage and a noncovalent linkage such as a linkageresulting from physicochemical interactions, such as adsorption, asdescribed above.

[0031] This anchorage of the macromolecule can be achieved directly on(or with) the surface, or indirectly, that is to say via a linkage suchas another molecule, especially another molecule with biologicalactivity. When the anchorage is achieved indirectly, the macromoleculecan be grafted chemically on the said linkage, or can interactphysicochemically with the said linkage, in particular when the saidintermediate linkage is a molecule with biological activity recognizingand interacting with the said macromolecule.

[0032] In one embodiment, the macromolecule and the said linkage areboth molecules with biological activity which interact, such as anantigen and an antibody respectively, complementary nucleic acids orlipids. In these cases, the noncovalent attachment of the macromoleculeconsists of a linkage of the type: antigen-antibody, ligand-receptor,hybridization between complementary nucleic acid fragments orhydrophobic or hydrophilic interaction between lipids.

[0033] Advantage is thus taken of the very high specificity and the veryhigh selectivity of certain biological reactions, especiallyantigen-antibody reactions, DNA or RNA hybridization reactions,interprotein reactions or avidin/streptavidin/biotin type reactions, aswell as reactions of ligands and their receptors.

[0034] Thus, in order to carry out the direct or indirect anchoring ofthe macromolecule on the surface S, it is possible to use a solidsurface having certain specificities. It is in particular possible touse certain pretreated surfaces which make it possible to attach certainproteins or DNA, whether modified or otherwise.

[0035] Such surfaces are commercially available (Covalink, Costar,Estapor, Bangs, Dynal for example) in various forms having at theirsurface COOH, NH₂ or OH groups for example.

[0036] It is, in this case, possible to functionalize the DNA with areactive group, for example an amine, and carry out a reaction withthese surfaces. However, these methods require specificfunctionalization of the DNA to be attached.

[0037] A technique allowing anchorage without prior treatment of the DNAhas also been described. This process consists in causing a freephosphate at the 5′ end of the DNA molecule to react with a secondaryamine of the surface (NH Covalink surface).

[0038] Anchoring by adsorption can be achieved by adsorption of the endof the molecule by controlling the surface charge by means of the pH,the ionic content of the medium or the application of an electricvoltage over the surface given the differences in adsorption between theends of the molecule and its middle part. According to the presentinvention, nonfunctionalized DNA molecules were thus anchored, by way ofexample, on surfaces coated with molecules ending with a vinyl or aminegroup such as polylysine molecules, or various surfaces such as glass,coated with silane type molecules ending with vinyl or amine groups oralternatively glass cover slips previously cleaned in an acid bath. Inthis latter case, the surface of the glass indeed has SiOH groups.

[0039] In all these cases, the pH range where the DNA is anchored ischosen to be between a state of complete adsorption and an absence ofadsorption, the latter being situated at a more basic pH. It isunderstood that this technique is very general and can be extended bypersons skilled in the art to numerous types of surfaces.

[0040] It is also possible to functionalize the DNA with a firstreactive group or a protein P₀ in order to cause it to react with asurface coated with a second reactive group or with a protein P₁, whichare capable of reacting specifically with each other respectively, thatis to say for example P₁ with P₀. The P₀/P₁ pair may be a pair of thetype: biotin/streptavidin (Zimmermann and Cox) or digoxigenin/antibodydirected against digoxigenin (anti-DIG) for example (Smith et al.,Science 258, 1122 (1992)).

[0041] Preferably, the anchoring surfaces will have a low fluorescencelevel so as not to interfere with the detection of the molecules aftertheir alignment, in particular if the detection is done by fluorescence.

[0042] According to the present invention, a solid support having, underthe reaction conditions, a surface having an affinity for only part ofthe macromolecule, the rest of the macromolecule remaining freely insolution, is preferably used.

[0043] In one embodiment, a solid support is used which has at thesurface at least one layer of an organic compound having, outside thelayer, an exposed group having an affinity for a type of molecule withbiological activity which may be the said molecule itself or a moleculerecognizing and/or interacting with it.

[0044] The support can therefore have a surface coated with a reactivegroup or with a molecule with biological activity.

[0045] By “affinity”, there should be understood here both a chemicalreactivity and an adsorption of any type, this under optional conditionsof attachment of the molecules onto the exposed group, modified orotherwise.

[0046] In one embodiment, the surface is essentially compact, that is tosay that it limits access by the macromolecule with biological activityto the inner layers and/or to the support, this in order to minimizenonspecific interactions.

[0047] It is also possible to use surfaces coated with a reactiveexposed group (for example NH₂, COOH, OH, CHO) or with a macromoleculewith biological activity (for example: proteins, such as streptavidin orantibodies, nucleic acids such as oligonucleotides, lipids,polysaccharides and derivatives thereof) which is capable of attachingan optionally modified part of the molecule.

[0048] Thus, surfaces coated with streptavidin or with an antibodyaccording to known processes (“Chemistry of Protein Conjugation andCross-linking”, S. C. Wong, CRC Press (1991)) are capable of attaching amacromolecule having, at a specific site, a biotin or an antigen.

[0049] Likewise, surfaces treated so as to have single-strandedoligonucleotides can serve in order to anchor on them DNAs/RNAs having acomplementary sequence.

[0050] Among the surfaces having an exposed reactive group, there may bementioned those on which the exposed group is a —COOH, —CHO, NH₂, —OHgroup, or a vinyl group containing a double bond —CH═CH₂ which is usedas it is or which can be activated so as to give especially —CHO, —COOH,—NH₂ OR OH groups.

[0051] The supports with highly specific surfaces according to thepresent invention can be obtained using various processes. There may bementioned by way of example:

[0052] (A) a layer of carbon-containing, optionally branched, polymer atleast 1 nm thick, having reactive groups as defined below and

[0053] (B) surfaces obtained by depositing or anchoring on a solidsupport one or more molecular layers; the latter can be obtained byforming successive layers attached through noncovalent linkages, asnon-limiting example, Langmuir-Blodgett films, or by molecular selfassembly, this allowing the formation of a layer attached by covalentlinkage.

[0054] In the first case, the surface can be obtained by polymerizationof at least one monomer generating at the surface of the polymer thesaid exposed group, or alternatively by partial depolymerization of thesurface of a polymer to generate the said exposed group, oralternatively by deposition of polymer.

[0055] In this process, the polymer formed has vinyl linkages such as apolyene derivative, especially surfaces of the synthetic rubber type,such as polybutadiene, polyisoprene or natural rubber.

[0056] In the second case, the highly specific surface contains:

[0057] on a support, a substantially monomolecular layer of an organiccompound of elongated structure having at least:

[0058] an attachment group having an affinity for the support, and

[0059] an exposed group having no or little affinity for the saidsupport and the said attachment group under attachment conditions, butoptionally having, after chemical modification following the attachment,an affinity for one type of biological molecule.

[0060] The attachment can first of all be of the noncovalent type,especially of the hydrophilic/hydrophilic and hydrophobic/hydrophobictype, as in Langmuir-Blodgett films (K. B. Blodgett, J. Am. Chem. Soc.57, 1007 (1935).

[0061] In this case, the exposed group or the attachment group will beeither hydrophilic or hydrophobic, especially alkyl or haloalkyl groupssuch as CH₃, CF₃, CHF₃, CH₂F, the other group being hydrophilic.

[0062] The attachment can also be of the covalent type, the attachmentgroup will, in this case, react chemically with the support.

[0063] Certain surfaces of similar structure have already been mentionedin the electronic field, especially when the attachments are covalent,L. Netzer and J. Sagiv, J. Am. Chem. Soc. 105, 674 (1983) and U.S. Pat.No. 4,539,061.

[0064] Among the attachment groups, there must be mentioned moreparticularly the groups of the metal alkoxide or semiconductor type, forexample silane, especially chlorosilane, silanol, methoxy- andethoxysilane, silazane, as well as phosphate, hydroxyl, hydrazide,hydrazine, amine, amide, diazonium, pyridine, sulfate, sulfonic,carboxylic, boronic, halogen, acid halide, aldehyde groups.

[0065] Most particularly, as attachment group, groups capable ofcross-reacting with an adjacent equivalent group, to give cross-linkageswill be preferably used; for example they will be derivatives of themetal alkoxide or semiconductor type, for example silane, especiallydichlorosilane, trichlorosilane, dimethoxysilane or diethoxysilane andtrimethoxy- or triethoxysilane.

[0066] The choice of the attachment group will obviously depend on thenature of the support; the silane-type groups are quite suitable forcovalent attachment on glass and silica.

[0067] As regards the exposed groups, irrespective of the surface, theywill be preferably chosen from ethylenic groups, acetylenic groups oraromatic radicals, primary, tertiary or secondary amines, esters,nitriles, aldehydes, halogens. But they may be most particularly thevinyl group; indeed, the latter can be either chemically modified afterattachment to give, for example, a carboxylic group or derivatives ofcarboxylic groups such as alcohol groups, aldehyde groups, ketonegroups, acidic groups, primary, secondary or tertiary amines, or to leadto a pH-dependent direct anchoring of the biological macromolecules suchas nucleic acids and proteins, without chemical modification of thesurface or of the macromolecules.

[0068] Preferably, the chains connecting the exposed group to theattachment group are chains carrying at least 1 carbon atom, preferablymore than 6 and in general from 3 to 30 carbon atoms.

[0069] As regards the support itself, the use of glass, surface-oxidizedsilicon, a polymer or gold with or without pretreatment of the surface,is generally preferred.

[0070] In the case of glass or silica, there can be used advantageouslythe known techniques for surface functionalization using silanederivatives, for example: Si—OH+Cl₃—Si—R—CH═CH₂ gives Si—O—Si—R—CH═CH₂,R consisting for example of (CH₂)₄. Such a reaction is known inliterature, with the use of ultrapure solvents. The reaction leads to alayer of molecules having their C═C end at the surface exposed to theoutside.

[0071] In the case of gold, this being optionally in the form of a thinlayer on a substrate, the known techniques for surface functionalizationuse thiol derivatives, for example: Au+HS—R—CH═CH₂ gives Au—S—R—CH═CH₂,R consisting for example of (CH₂)₄. Such a reaction is described inliquid medium and leads, like the preceding trichlorosilane-silicareaction, to a layer of molecules having their C═C end at the surfaceexposed to the outside.

[0072] Of course the term “support” encompasses both a single surfacesuch as a slide, but also particles, either silica powder or polymerbeads, and also ordinary forms such as a bar, a fiber or a structuredsupport, which can moreover be made magnetic, fluorescent or colored, asis known in various assay technologies.

[0073] Preferably, the support will be chosen so as to have no or littlefluorescence when the detection will be carried out by fluorescence.

[0074] The surfaces obtained according to methods (A) or (B) above have:

[0075] (i) a very low level of intrinsic fluorescence, when necessary, afluorescence background noise (with a typical surface area of 100×100μm) smaller than the fluorescence signal of a single molecule to bedetected;

[0076] (ii) the possibility of detecting isolated molecules with an S/Nratio independent of the number of molecules, which is possible byvirtue of various techniques with a high S/N ratio which are describedbelow and which are based on the identification of the presence of amacroscopic marker having a weak nonspecific interaction with thesurface.

[0077] The surfaces thus obtained are preferably coated with amacromolecule with biological activity chosen from:

[0078] proteins,

[0079] nucleic acids

[0080] lipids

[0081] polysaccharides and derivatives thereof.

[0082] Among the proteins, there should be mentioned antigens andantibodies, ligands, receptors, but also products of the avidin orstreptavidin type, as well as derivatives of these compounds.

[0083] Among the RNAs and DNAs, there should also be mentioned the α, βderivatives as well as the thio derivatives and mixed compounds such asPNAs.

[0084] It is also possible to attach mixed compounds such asglycopeptides and lipopolysaccharides for example, or alternativelyother elements such as viruses, cells in particular, or chemicalcompounds such as biotin.

[0085] The attachment of the biological macromolecules may be covalentor noncovalent, for example by adsorption, hydrogen bonds, hydrophobic,ionic interactions for example, in which case cross-linking can beadvantageously carried out in the molecules grafted by known methods(“Chemistry of Protein Conjugation and Cross-linking”, S. C. Wong, CRCPress (1991)) and this in order to enhance their cohesion.

[0086] As mentioned above, it is possible to have an exposed group whichallows direct reaction with molecules with biological activity, but itis also possible to envisage that the exposed group is treated, afterattachment, so as to be converted, as indicated above, to a hydroxyl,amine, alcohol, aldehyde, ketone, COOH radical or a derivative of thesegroups before attachment of the biological molecule.

[0087] When such groups were exposed, techniques for attachment ofproteins and/or of DNA for example are known, they are indeed reactionsimplemented for surfaces which are already used for biological analysis,especially for Costar surfaces, Nunc surfaces or microbeads such asEstapor, Bang and Dynal for example, on which molecules of biologicalinterest, DNA, RNA, PNA, proteins or antibodies for example, areanchored.

[0088] In the case where the exposed group is a —CH═CH₂ radical which iscalled hereinafter “surface C═C” or “surface with ethylenic bond”, nodocument exists which mentions direct anchoring, in particular of DNA orof proteins.

[0089] Within the framework of the present invention, it has beendemonstrated that these surfaces have a highly pH-dependent reactivity.This characteristic makes it possible to anchor the nucleic acids or theproteins using pH regions and often with a reaction rate which can becontrolled by the pH.

[0090] The anchoring of DNA can be carried out by its end onto a surfacehaving groups with ethylenic double bonds, by bringing the DNA intocontact with the surface at a pH of less than 8.

[0091] In particular, the reaction is carried out at a pH of between 5and 6, and is then stopped at pH 8.

[0092] Thus, for DNA at pH 5.5, the anchoring reaction is complete inone hour (if it is not limited by diffusion) and occurs via the ends. AtpH 8 on the other hand, the attachment is very low (reaction rate of 5to 6 orders of magnitude smaller). This pH dependent attachment effectspecific for the ends, is an improvement compared with the othersurfaces which require functionalization of the DNA (biotin, DIG, NHS,and the like) or specific reagents (carbodiimide, dimethyl pimelidate)which form a peptide or phosphorimide linkage between —NH₂ and —COOH or—POOH.

[0093] It is also possible to carry out the anchoring of DNA byadsorption of its ends alone onto a surface coated with polylysine or asilane group ending with an amine group.

[0094] In order to carry out the anchoring of the DNA by its end on asurface coated with an amine group, the DNA is brought into contact withthe surface at a pH of between 8 and 10.

[0095] Likewise, it is possible to carry out the anchoring of DNA by itsend onto a glass surface treated beforehand in an acid bath, by bringingthe DNA into contact with the said surface at a pH of between 5 and 8.

[0096] It goes without saying that the present invention involves, inthe same spirit, the optionally pH-dependent attachment of allmacromolecules of biological interest.

[0097] Likewise, these surfaces can anchor proteins directly (protein A,anti-DIG, antibodies, streptavidin and the like). It has been observedthat (i) the activity of the molecule can be preserved and (ii) that thereactivity of the prepared surface (initially C═C) is completelyovershadowed in favor of the sole reactivity of the molecule ofinterest. It is therefore possible, starting with a relatively highinitial reactivity, to pass to a surface having a very highly specificreactivity, for example that of specific sites on a protein.

[0098] By anchoring a specific antibody on the surface (for exampleanti-DIG), a surface is created whose reactivity is limited to theantigen (for example the DIG group). This indicates that the initialchemical groups are all occulted by the antibodies grafted.

[0099] It is also possible to graft onto the reactive (chemically orbiochemically) surfaces other molecules with biological activity,especially viruses or other components: membranes, membrane receptors,polysaccharides, PNA, in particular.

[0100] It is also possible to attach the product of a reaction ofbiological interest (for example PCR) onto the prepared surfaces.

[0101] The process according to the present invention allows thedetection and/or the quantification of biological molecules, but alsothe measurement of intramolecular distance, the separation of certainbiological molecules, especially a sample using antigen/antibody and/orDNA/RNA coupling techniques.

[0102] In particular, the subject of the present invention is a processfor detecting a macromolecule, consisting of a DNA sequence or a proteinin a sample, according to the present invention, characterized in that:

[0103] the sample corresponding to solvent A, in which the saidmacromolecule is in solution, is brought into contact with the surfaceof the support under conditions for forming a DNA/DNA, DNA/RNA hybrid orfor forming the protein/protein reaction product,

[0104] the hybrid or a macromolecule for labeling the hybrid or thereaction product being anchored in one part, the remainder being free insolution, it is stretched by the movement of the meniscus created by thecontact between the solvent and the surface in order to orientate thehybrids or the said labeling macromolecules and the measurement or theobservation of the hybrids or of the said labeling macromolecules thusorientated is carried out.

[0105] Advantageously, the attached DNA and the DNA of the sample arecolored differently and after stretching, the position of thecomplementary sequence relative to the end of the sample DNA ismeasured.

[0106] Appropriately, the ELISA or FISH detection methods can be used.

[0107] The DNA sample may be the product or the substrate of a DNAenzymatic amplification such as PCR, that is to say that theamplification of the DNA can be carried out once it has been anchoredand aligned according to the process of the invention or before itsanchoring or its alignment.

[0108] The passage of the meniscus, by stretching the moleculeslinearly, in the form of rods, renders them more easily detectable ifthey are labeled. Moreover, these elongated molecules are stable to theopen air and can be observed even after several months, without showingapparent degradation.

[0109] During a rehydration, the DNA molecules can remain adsorbed andelongated. Furthermore, it is possible to carry out a hybridization onthe elongated molecule.

[0110] Furthermore, exhibiting a signal which is correlated and ofuniform orientation by virtue of their stretching, these molecules aredistinct from the surrounding noise. It is therefore easy to ignore thedusts, the inhomogeneities, which have no special spatial correlation.The alignment is also important because in solution, the molecules inthe form of a random cole fluctuate thermally, thereby causing very highvariations in their fluorescence signal gathered preferably with a smalldepth of field and limits their observation. The present inventiontherefore allows the observation of isolated molecules with a very highsignal to noise (S/N) ratio.

[0111] It is remarkable that this ratio is independent of the number ofmolecules anchored. The S/N ratio posed by the detection of a moleculeis the same as that for 10,000. Furthermore, this stretching techniquemakes it possible to easily discriminate between molecules of varyinglengths.

[0112] It is advantageously possible to proceed to the following stagesin order to further improve the S/N ratio:

[0113] The molecule being stationary, its fluorescence signal can beintegrated.

[0114] Microscopic observation presents a reduced field (typically 100μm×100 μm with a ×100 immersion lens, N.A.=1.25). For a 1 cm² sample,scanning can be carried out, or it is possible to envisage the use oflower magnification lenses (×10 or ×20) but with a high numericalaperture.

[0115] The rods being always parallel, it is possible to envisage anoptical spatial filtration method in order further to increase the S/Nratio.

[0116] Other global fluorescence methods can be envisaged such as thosedescribed in European Patent Application EP 103426.

[0117] The linearization of the molecules is observed both within theframework of a physicochemical anchoring and in the case ofimmunological type linkages (DIG/anti-DIG).

[0118] Once the surface is in the open air, the DNA molecules are stable(they maintain their integrity even after several weeks) andfluorescent. This property can be advantageously used in order to deferthe anchoring stage and the locating/counting stage for the moleculesanchored, if this detection is done for example, but without beinglimited thereto, by fluorescence microscopy. Such a use is covered bythe present invention.

[0119] A double (or multi) fluorescence technique can possibly be usedto improve the S/N ratio or to detect a double functionality.

[0120] The stretched molecules can be revealed by various enzymologicalmethods on others, such as fluorescence, or the use of radioactive ornonradioactive probes. Their detection can be achieved by measuring aglobal signal (for example the fluorescence) or by individualobservation of the molecules by optical fluorescence microscopy,electron microscopy, local probe methods (STM, AFM and the like).

[0121] Thus in general, the present invention allows the detection,separation and/or assay of a molecule in a sample, by a processcharacterized in that a surface capable of specifically attaching thesaid molecule is used, and in that the detection, separation or assayare carried out using a reagent, fluorescent or otherwise, which detectsthe presence of the attached molecule.

[0122] Among the reagents, there are fluorescent reagents andnonfluorescent reagents.

[0123] The fluorescent reagents contain fluorescent molecules,advantageously chosen to be long molecules of size greater than 0.1 μmand reacting specifically, directly or indirectly, with the pretreatedsurfaces. For example, but with no limitation being implied, adouble-stranded DNA molecule stained by means of fluorescent probes(ethidium bromide, YOYO, fluorescent nucleotides and the like) capableof anchoring directly via one or more ends on a surface optionallyhaving a vinyl or amine type group and the like, especially by ajudicious choice of the pH or of the ionic content of the medium or byapplication of an electric voltage over the surface.

[0124] It is also possible to use a special functionalization of themolecule (DIG, biotin and the like) in order to anchor it at one or morepoints on a surface having complementary sites (anti-DIG, streptavidinand the like).

[0125] Nonfluorescent reagents allowing the detection of moleculespreviously aligned according to the present invention may consistespecially of beads or microparticles anchored via another moleculeattached specifically, directly or indirectly, to the aligned moleculeand having only a weak nonspecific interaction with the surface.

[0126] For example, there may be mentioned Dynal beads coated withstreptavidin permitting anchoring on biotinylated DNA aligned accordingto the present invention.

[0127] Depending on whether the desired molecule is detected directly byfluorescence or indirectly by means of the above reagents, the detectionwill be described as “direct detection” or “flag detection”.

[0128] In order to limit the problems associated with too slow reactiontimes, the diffusion times of the reagents towards the surface can beadvantageously reduced using small reaction volumes. For example, butwith no limitation being implied, by carrying out the reaction in avolume of a few microliters determined by the space between two surfacesof which one is treated so as to have reactive sites and the other isinert or treated so as not to have reactive sites, under the reactionconditions.

[0129] The detection of the number of aligned molecules can be carriedout on a small number of molecules (typically 1 to 1000), by a low-noisemacroscopic physical test requiring neither electron microscope norradioactivity nor necessarily PCR.

[0130] The alignment and detection processes according to the presentinvention are capable of being carried out by persons having onlylimited laboratory experience.

[0131] The specificity of certain biological reactions may be limited.Thus, within the framework of the hybridization, the hybrids may beimperfect (reactions with other sites) while having a reduced number ofpairing and therefore a lower quality of binding. The present inventionalso covers the possible use of a stage for testing the quality of thebonds obtained. This test makes it possible to dissociate the productsweakly and nonspecifically paired by adsorption, hydrophobic forces,imperfect hydrogen bonds, imperfect hybridization, in particular.

[0132] Accordingly, the invention also relates, in a detection or assayprocess as described above, to a process where the product of thereaction between the molecule with biological activity and the samplemolecule is subjected to a stress in order to destroy the mismatchesbefore the detection.

[0133] This process offers, in addition to the possibility of destroyingthe mismatched pairs, the possibility of orientating the products of thecoupling, which facilitates the measurements or the observations.

[0134] It is thus possible to apply to the surfaces, after attachment ofthe complementary elements, a stress which may consist of the single orcombined use of:

[0135] centrifugation,

[0136] gradient of magnetic field applied to the nonfluorescent reagentstaken, in this case, to include magnetizable or magnetic microbeads,

[0137] stirring,

[0138] liquid flow,

[0139] meniscus passage,

[0140] electrophoresis

[0141] temperature variation, and/or temperature gradient.

[0142] The number of systems to have remained intact or to have beendestroyed is then determined by the low-noise detection techniquesdescribed above.

[0143] The alignment and detection techniques described is according tothe present invention can be used for numerous applications among which,but with no limitation being implied:

[0144] the identification of one or more elements of DNA or RNA sequencewhich can be advantageously used for the diagnosis of pathogens or thephysical map of a genome. In particular, the techniques described abovemake it possible to obtain a physical map directly on genomic DNAwithout the intermediate use of a cloning stage. It is understood thatthe combed molecule having been stretched relative to itscrystallographic lengths, relative measurements are carried out. It isthus possible to measure the size of the DNA fragments and the distancebetween fragments, with a resolution of the order of 200 nm by opticalmethods or of the order of 1 nm by the use of near field methods such asAFM or STM in order to visualize and measure the distance between probeson the aligned DNA.

[0145] This naturally leads to:

[0146] 1) the detection of deletions, additions or translocations ofgenomic sequences, in particular in the diagnosis of genetic diseases(for example Duchesne's myopathy);

[0147] 2) the identification of promoters of various genes by measuringthe distance between the regulatory sequences and those expressed;

[0148] 3) the localization of regulatory proteins by identifying theirposition along the DNA or the position of their target sequence;

[0149] 4) the partial or complete sequencing by measuring the distanceusing near field microscopy (for example AFM or STM) between hybridizedprobes belonging to a base oligonucleotide of given length;

[0150] the enzymatic amplification in situ on aligned DNAs;

[0151] the improvement of the sensitivity of ELISA techniques with thepossibility of detecting a small number (possibly less than 1000) ofimmunological reactions.

[0152] Thus, physical mapping can be carried out directly on a genomicDNA without the intermediate use of a cloning stage. The genomic DNA isextracted, purified, optionally cleaved with one or more restrictionenzymes and then combed on surfaces according to the process of thepresent invention.

[0153] The position and size of the desired gene on the genomic DNA arethen determined by hybridization with probes specific for the said gene,especially extracted from parts of the complementary DNA (cDNA) of theproduct of the said desired gene.

[0154] Similarly, by hybridizing a genomic DNA combed, then denaturedwith total cDNA labeled by fluorescence or any other marker allowing thehybrid to be localized, the position, size and number of exons of thegene in question are identified and its size and its geneticorganization (exons, introns, regulatory sequences) are deducedtherefrom.

[0155] The position of the gene having been determined as describedabove or being known, it is then possible to identify, by hybridization,the flanking sequences of the gene. For that, the procedure isadvantageously carried out by hybridization with labeled probes,obtained for example from an oligonucloetide library, in order toidentify two or more probes which hybridize on either side of the gene.

[0156] From this determination, it is then possible, by enzymaticamplification techniques, for example in situ PCR (Nuovo G. J. PCR insitu hybridization: protocols and applications, Raven Press (1992)) toamplify the fragment delimited by the flanking probes which can serve asprimers for the reaction, which fragment may contain the desired genewith its regulatory regions which may be tissue- or development-specificand which can then be isolated and purified.

[0157] The procedure can also be carried out by in situ polymerizationon primers extracted from the cDNA of the gene in question in order toextract DNA fragments complementary to the flanking regions of the geneas mentioned by Mortimer et al. (Yeast 5, 321, 1989). These fragmentscan then serve in the preparation of primers for a process of enzymaticamplification of the gene and of its flanking sequences.

[0158] The methods cited by A. Thierry and B. Dujon (Nucl. Acid Research20 5625 (1992)) for inserting, by homologous recombination or randomly,known endonuclease-specific sites into a genomic DNA or a genomic DNAfragment, may also be used. The combing of this DNA allows theidentification of the gene of interest and of the specific sitesinserted, by the in situ hybridization methods described above. Fromthis identification and preferably, if the sites of interest are regionsof interest which are close to the gene, they will be used as primer fora reaction of enzymatic amplification (in situ and the like) of the genein question and of its flanking sequences.

[0159] The amplification of the desired gene then proceeds using knownenzymatic amplification techniques such as PCR on the amplified fragmentas described above, using primers which can be reached by the exonsconstituting the cDNA, or primers corresponding to flanking sequences.

[0160] By the combing of genomic DNA and the like, it is also possibleto determine, by hybridization, the presence or the absence ofregulatory sequence of a specific proximal gene, from which the possiblefamilies of proteins for regulating this gene (for example:helix-loop-helix, zinc-finger, leucine-zipper) will be determined.

[0161] The specific reactions between particular DNA/RNA/PNA sequencesand another molecule (DNA, RNA, protein) can occur before or afteraligning the molecules according to the prevent invention.

[0162] Thus, in the field of genetic diagnosis and physical mapping, theknown methods of FISH (Pinkel et al., Proc. Nat. Acad. Sci. USA 83, 2934(1986)) are advantageously used to hybridize single-strandedoligonucleotides labeled with DNA first aligned, and then denatured. Therevealing of the hybrids will be carried out using known techniques(fluorescence, microbeads and the like) with a resolution in themeavurement of the distances ranging from 0.2 μm (optically) to 1 nm (bynear field microscopy; AFM, STM and the like).

[0163] Alternatively, it is possible to first hybridize fluorescentlylabelled DNAs to single-stranded DNA in solution, and then to align thisconstruct by action of the meniscus after having converted it todouble-stranded DNA and anchored it on an appropriate surface.

[0164] It is also possible to use the present invention for detectingthe prevence of a pathogen. By way of example, the procedure can becarried out in two different ways depending on whether the recognitionreaction (hybridization, attachment of proteins) occurs before or afteralignnent by the meniscus.

[0165] Thus, by way of example, one or several oligonucleotide probesare anchored in one or more regions of a surface. The hybridization ofthe potentially pathogenic DNA is carried out in situ under stringentconditions so as to anchor only the hybridized molecules. Theirdetection and quantification is carried out after alignnent by themeniscus according to the present invention.

[0166] Alternatively, the potentially pathogenic DNA is first aligned,then denatured and hybridized with an oligonucleotide probe understringent conditions. The detection of the hybrid is then carried out byknown methods, especially by the FISH method, as described above.

[0167] Similarly, it is possible to detect the presence (or the absence)of a small numher of molecules, such as proteins, lipids, sugars orantigens. A minor modification of the ELISA techniques will beadvantageously carried out, the usual detection method being replaced bythe detection of a fluorescent molecule aligned according to the presentinvention and coupled to one of the reagents of the ELISA reaction.

[0168] Moreover, as mentioned by R. R. Allan et al. (U.S. Pat No.84,114), genetic mapping can be carried out by measuring the size of theDNA fragments. Now, the novel techniques for stretching moleculesdescribed above (stretching by meniscus) allows the length of thestretched molecules to be measured, and this on a very small sample (afew thousandths of molecules).

[0169] It is for example possible, but with no limitation being implied,to carry out the procedure in the following manner:

[0170] A DNA sample is fragnented (by means of restriction enzymes)stained with a fluorophore and then anchored on a surface. The moleculesare then stretched by the meniscus and the size of the stretchedfragments is determined by optical fluorescence microscopy with aresolution and a maximum size of the order of 1000 bp (0.3 μm).

[0171] For this purpose, but also if it is desired to align very longmolecules (≧10 μm), known techniques will be advantageously used inorder to limit the degradation of long macromolecules during theirhandling (by hydrodynamic shearing).

[0172] Thus, as mentioned by D. C. Schwartz, condensation of themolecules will be advantageously carried out by means of a condensingagent (for example spermine or an alcohol) during their handling.Optionally, their decondensing will occur during contact between thesolvent A and the anchoring surface S.

[0173] In order to reduce the degradation of the macromolecules duringstretching by the meniscus, meniscus translation techniques. will beused which minimize hydrodynamic shearing. For example, but with nolimitation being implied, by very slowly removing (≦200 μ/sec) thesurface S from a substantial volume (≧100 μl) of the solvent A.

[0174] The subject of the present invention is also a surface having oneor more types of aligned macromolecules obtained according to thepresent invention. In particular, it is possible to obtain a surface ora stack of surfaces having anisotropic optical or electrical properties.

[0175] The subject of the present invention is also a process foraligning and detecting DNA in which the DNA is stretched by an aligningprocess according to the invention, then denatured and hybridized withspecific probes in order to determine the position or the size of one ormore specific sequences.

[0176] The subject of the present invention is also a process for thephysical mapping of a gene on a genomic DNA in which the DNA is alignedor detected according to a process of the invention.

[0177] In particular, the position and the size of the desired gene onthe genomic DNA are determined by hybridization with probes specific forthe said gene to be mapped.

[0178] A subject of the present invention is also

[0179] a kit useful for carrying out a mapping process according to theinvention, consisting of total genomic DNA from a reference host,

[0180] a support having a surface permitting the anchoring and thealignment of the patient's DNA in accordance with the process of theinvention

[0181] probes specific for the gene(s) to be mapped and reagents for thehybridization and the detection of the DNA.

[0182] The subject of the present invention is also a process foraligning and detecting DNA in which the DNA is stretched, then denaturedand hybridized with specific probes in order to determine the prevenceor the absence of one or more DNA sequences in the said aligned DNA.

[0183] The prevent invention allows the implementation of a process forthe diagnosis of a pathology related to the presence or the absence of aDNA sequence specific for the pathology in which an alignment processaccording to the invention is used.

[0184] The subject of the prevent invention is also a kit useful forcarrying out a diagnostic process according to the invention,characterized in that it contains a support whose surface permits theanchoring and the alignsent of the patient's DNA according to a processof the invention, probes specific for the gene involved in the soughtpathology and reagents for the hybridization and the detection of theDNA.

[0185] The subject of the prevent invention is also a kit useful forcarrying out a diagnostic process according to the invention,characterized in that it contains a support whose surface has probesspecific for the gene involved in a pathology, in particular optionallylabeled pathogenic DNA, which are aligned according to the process ofthe present invention and optionally denatured; the reagents forpreparing and labeling the patient's DNA for its hybridization (forexample photobiotin, nick translation or random priming kit) andreagents for the hybridization and the detection of the DNA according tothe in situ hybridization techniques as described above.

[0186] It is understood that combed probes relating to differentpathogens may be prevent on different supports or on the same support.The identification of the corresponding pathogen can be carried outafter hybridization, either spatially (the different probes arespatially separated for example by photochemical anchoring before theircombing) or by a difference in the fluorescence spectrum of thedifferent hybrids, resulting from a prior differential labeling of theprobes.

[0187] Finally, the subject of the present invention is a process forpreparing a gene in which the position of the said gene on the genomicDNA aligned by the process according to the invention is identified bymeans of a probe specific for the said gene, the sequence of the saidgene and optionally its flanking sequences are amplified by enzymaticamplification, in particular by in situ PCR.

[0188] The present invention therefore makes it possible to carry out aprocess for replacing a gene in the genome of an eukaryotic cell bytargeted insertion of a foreign gene by means of a vector containing thesaid foreign gene prepared according to the above gene preparationprocess.

[0189] The targeted insertion can be carried out according to thetechniques described in WO90/11354 by transfecting eukaryotic cells witha vector containing the said foreign DNA to be inserted flanked by twogenomic sequences which are contiguous to the devired site of insertionin the recipient gene. The insert DNA may contain either a codingsequence, or a regulatory sequence. The flanking sequences are chosen soas to allow, by homologous recombination, depending on the case, eitherthe expression of the coding sequence of the insert DNA under thecontrol of the regulatory sequences of the recipient gene, or theexpression of a coding sequence of the recipient gene under the controlof a regulatory sequence of the insert DNA.

[0190] The genomic genes and the cDNAs obtained using the process forlocalizing genes according to the invention can be inserted intoexpression vectors capable of being inserted into a prokaryotic,eukaryotic or viral host cell. The derived proteins, polypeptides andpeptides are included in the present invention.

[0191] The following description is made with reference to theaccompanying figures in which:

[0192]FIG. 1 schematically represents the detection of a pathogen in afluorescent DNA molecule by hybridization with an anchor molecule;

[0193]FIG. 2 schematically represents genetic mapping by extension ofDNA and the use of a marker DNA;

[0194]FIG. 3 schematically represents the detection of an immunologicalreaction (ELISA) by means of a “flag” molecule: a fluorescent DNA usedas reaction marker;

[0195]FIG. 4 is a fluorescence micrograph showing the extension of λphage DNA by the progression of the meniscus, DNA molecules in solutionstretched by the evaporation flow parallel to the meniscus can be seenon the left, DNA molecules in the open air after being stretchedperpendicularly to the meniscus can be seen on the right;

[0196] FIGS. 5(a) and 5(b) are fluorescence micrographs showing,respectively, a DNA labeled with digogixenine [sic] (DIG) on a surfacecoated with anti-DIG and stretched by the meniscus, and, as control, anunlabeled DNA on an anti-DIG surface; the very high specificity of thesurfaces and the absence of nonspecific anchoring will be noted;

[0197]FIG. 6 represents the schematic representation of the spread ofthe DNA by passage of the meniscus. The DNA in solution is anchored on atreated surface. The DNA solution is covered with an untreated roundcover slip;

[0198]FIG. 7 represents histograms of the length of the combed λ DNAmolecules on glass surfaces:

[0199] a) coated with silane molecules ending with an amine group,

[0200] b) coated with polylysine,

[0201] c) cleaned in a hydrogen peroxide/sulfuric acid mixture;

[0202]FIG. 8 represents combed DNA molecules on glass surfaces coatedwith polylysine. It can be noted that the molecules attached by theirtwo ends form loops;

[0203]FIG. 9 represents YACs combed by removal of a treated cover slipin a solution of these molecules;

[0204]FIG. 10 shows the identification of the presence and the size of acosmid on a YAC by in situ hybridization.

[0205] In the “diagnostic” mode, the probes (the “anchors”) possess areactive group (DIG, biotin and the like) capable of anchoringspecifically on a surface according to the present invention (having forexample as anchoring site an anti-DIG antibody or streptavidin). Thedetection of the anchoring reaction can be carried out directly bydetection of the fluorescence of the DNA molecule stained by fluorescentmolecules (ethidium bromide, YOYO, fluorescent nucleotides) (FIG. 1). Itcan also be carried out indirectly by detection of a “flag molecule”: areagent capable of attaching to the DNA/RNA molecule (for example byhybridization, protein-DNA interaction and the like), but having noaffinity for the anchoring sites of the probe.

[0206] In the “mapping” mode, in situ hybridization techniques (FISH)can be used. It is also possible to envisage other techniques, forexample by hybridizing in solution DNA with probes having fluorescentreagents according to the present invention. The detection of theposition of the probes is carried out after aligning the moleculeaccording to the prevent invention.

EXAMPLE 1

[0207] Materials and Methods

[0208] The λ DNA and the monoclonal antibody (anti-DIG) are obtainedfrom Boehringer-Mannheim. The trichlorosilanes are obtained fromRoth-Sochiel. The fluorescent nucleic probes (YOYO1, YOYO3 and POPO1)are obtained from Molecular Probes. The ultraclean glass cover slips areobtained from Erie Scientific ((ESCO) cover slips). The magneticparticles are obtained from Dynal. The microscope is a Diaphot invertedmicroscope from NIKKON, equipped with a Xenon lamp for epifluorescenceand a Hamamatsu intensified CCD camera for the visualization.

[0209] Surface Treatment

[0210] Glass cover slips are cleaned for one hour by UV irradiationunder an oxygen atmosphere (by formation of ozone). They are thenimmediately placed in a desiscator previously purged of traces of waterby an argon stream. A volume of about 100 to 500 μl of the appropriatetrichlorosilane (H₂C═CH—(CH₂)_(N)—SiCl₃)is introduced into thedesiccator, from which the surfaces are removed after about 12 hours(n=6) or 1 hour (n=1). Upon taking out, the surfaces are clean andnonwetting.

[0211] The functional groups of these double bond surfaces (H₂C═CH—) canbe converted to carboxyl groups (—COOH) by soaking the treated coverslips, as described above, for about ten minutes in a solution of 25 mgKMnO₄, 750 mg NaIO₄ in 1 l of water, then by rinsing them three times inultrapure water.

[0212] The cover slips thus functionalized can react with proteins. Avolume of 300 μl of an aqueous solution (20 μg/ml) of proteins (proteinA, streptavidin and the like) is deposited on a cover slipfunctionalized with a (H₂C═CH—) group. This cover slip is incubated forabout two hours at room temperature, then rinsed three times inultrapure water. The surfaces thus treated are clean and wetting. Thesurfaces treated with protein A can then react with an antibody, forexample an anti-DIG antibody, by incubating in a solution of 20 μg/ml ofantibody.

[0213] Moreover, on the surfaces having carboxyl groups, it is possibleto graft oligonucleotides having an amine end (—NH₂), 200 μl of asolution of MES (50 mM, pH 55), carbodiimide (1 mg/ml) and 5 μl ofamino-oligo-nucleotide (10 pmol/140 μl) are deposited on a carboxylatedsurface and incubated for about 8 hours at room temperature. The coverslip is finally rinsed three times in NaOH (0.4 M) and then four timesin ultrapure water. The cover slips thus prepared can hybridize DNAscomplementary to the anchored oligonucleotide.

[0214] Anchorina of Native DNA on a Double Bond Surface

[0215] A drop of 2 μl of a fluorescence-labeled λ DNA (YOYO1, POPO1 orYOYO3, but withno specific end labelling) of varying concentration andin different buffers (total number of molecules <10⁷) is deposited on apretreated cover slip (C═C) and covered with an untreated glass coverslip (diameter 18 mm). The preparation is incubated for about 1 hour atroom temperature in an atmosphere saturated with water vapor. In a 0.05M MES buffer (pH=5.5), a virtually general anchoring of the DNAmolecules is observed. In contrast, in a 0.01 M Tris buffer (pH=8),there is practically no anchored molecule (ratio >10⁶). This dependencecan make it possible to control the activation/deactivation of surfaces(with respect to DNA) via the pH.

[0216] The action of the meniscus on the molecule is limited to itsimmediate vicinity. The part of the molecule in solution in front,of themeniscus fluctuates freely and the part left stuck on the surface behindthe meniscus is insensitive to a change in the direction of themeniscus. The extension rate of the molecule is therefore uniform andindependent of its size.

[0217] Alignment and Detection of the Anchoring by the Action of theMeniscus

[0218] By transferring the preceding preparation to a dry atmosphere,the solution, upon evaporating, will stretch the DNA molecules anchoredon the surface, perpendicularly to the meniscus. The capillary force onthe DNA molecule (a few tens of picoNewtons) is indeed sufficient tocompletely stretch the molecule (greater than the entropic elasticityforces), but too weak to break the bond between the end of the moleculeand the treated surface. The DNA having been fluorescence labeled, thestretched molecules (total length about 22 μm) can be individually andeasily observed. The anchoring between the surface and the DNA beinglimited to the ends, it is possible to stretch either DNA of λ phage, ofYAC or of E. coli (total length greater than 400 μm). This DNApreparation, stretched, fluorescent and in the open air, is stable forseveral days and can be observed in a nondestructive manner, byepifluorescence (Nikkon Diaphot inverted microscope with a ×100 lens,O.N.: 1.25).

[0219] Specific Anchoring and Detection

[0220] By treating the surfaces as described above with a specificmonoclonal antibody, it is possible to control their specificity veryprecisely. Thus, the specificity of anti-DIG treated surfaces was testedin relation to λ DNA hybridized with an oligonucleotide complementary toone of the Cos ends and possessing a digoxigenin group (DIG) and inrelation to nonhybridized DNA. In the first case, a virtually generalextension of the anchored molecules, by the action of the meniscus, wasobserved. In the second case, only a few anchored DNA molecules (<10)were observed in the whole sample. It is therefore estimated that thespecificity of the method according to the invention is greater than106.

[0221] λ DNAs were also hybridized with oligonucleotides complementaryto one of the COS ends and attached to carboxylated surfaces, asdescribed above. The hybridization conditions (pure water at 40° C.)were not stringent, because under stringent conditions (high salinity)the fluorescence of the YOYO1 probes disappears and the hybridized DNAscannot be seen. It was also possible to align the DNAs thus hybridizedby passage of the meniscus.

[0222] Sensitivity of the Detection

[0223] In order to determine the sensitivity of the detection method byextension of the meniscus, 2.5 μl drops of a solution of λ DNA in 0.05 MMES (pH=5.5) containing a total of 10⁵, 10⁴ and 1000 molecules, weredeposited on double bond surfaces. The anchoring and the alignment arecarried out as described above. The cover slips are then observed byepifluorescence microscopy to determine the density of combed molecules.The latter indeed corresponds to that estimated: about 4-6 DNA moleculesper field of vision (100 μm×100 μm) for a total of 10⁵ DNA molecules.For the lowest concentration, it was possible to observe about tenmolecules extended by the action of the meniscus. This number isessentially limited by the large number of fields of vision required tocover the whole sample (about 25,000), which makes a manual searchdifficult, but it can be advantageously carried out automatically oralso with a weaker lens, but with a larger field. In conclusion, thesensitivity of the method according to the invention allows detectionand individual counting of less than 1000 DNA molecules.

[0224] Dependence of the Stretching on the Surface Treatment

[0225] The histogram of the lengths of λ DNA grafted on differentsurfaces and then aligned by passage of the meniscus shows a welldefined peak but which is different for the different surfaces. Thus, onsurfaces coated with a silane which end with a vinyl group, the DNA isstretched up to about 22 μm (see above) for surfaces silanized with anamine group (—NH₂), the histogram has a peak at 21 μm (FIG. 7(a)) and onclean glass at about 18.5 μm (FIG. 7(c)).

[0226] The stretching therefore depends on the surface treatment.

EXAMPLE 2

[0227] Combing of DNA Molecules on Different Surfaces

[0228] The molecular combing of DNA on glass surfaces treated in variousways was observed. Advantage is taken of the difference in adsorptionbetween the ends of the molecule and the rest of the molecule. Byadsorbing positively charged polyners onto a glass surface, adsorptionof negatively charged DNA molecules is enhanced, however when thischarge is large, the DNA molecule is stuck over its entire length andthe combing is impossible. However it is possible to modify the chargeof the polymers adsorbed on the glass by modifying the pH conditions,indeed, the positive charges are carried for example by the NN2 groupswhich pass to the protonated state NE₃ _(⁺) for a pH below the pK of thecorresponding base. In basic pH, the charges disappear and the surfaceno longer attracts DNA. By finely controlling the pH, it was observedthat the DNA molecules in solution passed from a state where they arecompletely stuck to the surface to an intermediate phase where they areanchored only by their ends and then to a phase where the surface nolonger has affinity for the DNA. In the intermediate phase, molecularcombing can be carried out.

[0229] Surfaces coated with a silane ending with an NH₂ group werestudied for which there is observed complete sticking at pH<8, andcombing for 8.5<pH<9.5. The number of combed molecules is maximum atpH=8.5 it is divided by 2 at pH=9 and by 4 at pH=9.5. Also the relativeextension on this surface which corresponds to 1.26 was determined ascan be seen in histogram 2 of FIG. 7 which represents histograms of thelength of the combed λ DNA molecules on glass surfaces:

[0230] a) coated with silane ending with an amine group,

[0231] b) coated with polylysine,

[0232] c) cleaned in a hydrogen peroxide/sulfuric acid mixture.

[0233] Surfaces coated with polylysine were also examined and found toexhibit similar attachment characteristics as regards the pH: combingregion at 8,5 and exhibiting a shorter relative extension: 1.08. Atypical example can be obtained in FIG. 8 which represents combed DNAmolecules on glass surfaces coated with polylysine. It can be observedthat the molecules attached by their two ends form loops.

[0234] Finally, the same behavior was found on glass surfaces freshlycleaned in a hydrogen peroxide/concentrated sulfuric acid mixture. Thesesurfaces are highly wetting and become rapidly contaminated; however, acombing region was observed between 5.5<pH<7.4 whereas the region ofstrong adsorption is situated at pH=4.5. The relative extension of themolecules corresponds to 1.12.

EXAMPLE 3

[0235] Uniform and Directional Alignment of YAC

[0236] 1 μg of YAC previously stained in its agarose plug by means of aYOYO1 fluorescent probe is heated to 68° C., agarased and then dilutedin 10 ml of MES (50 mM pH 5.5). Two silanized cover slips (C═C surfaces)are incubated for ≈1.5 h in this solution and then removed at about 170μm/sec. The YAC molecules are all aligned parallel to the direction ofremoval of the cover slips (FIG. 9). The integrity of the molecules thusaligned is better than by evaporation after deposition between two coverslips.

[0237] Hybridization of a Cosmid with a Combed YAC

[0238] A YAC stained as previously described is anchored on a C═Csurface (between two cover slips) and then aligned by the meniscus,during evaporation of the solution. The probes (cosmids) are labeled byincorporation of a biotinylated nucleotide by the random primingtechnique. The labeled probes (100 ng) and 5 μg of sonicated salmonsperm DNA (≈500 bps) are purified by precipitation in Na-acetate andethanol, and then denatured in formamide.

[0239] The combed YACs are denatured between two cover slips with 120 μlof denaturing solution (70% formamide, 2×SSC) on a hotplate at 80° C.for 3 minutes. The previously denatured probes (20 ng) are deposited onthe cover slip in a hybridization solution (55% formamide, 2×SSC, 10%dextran sulfate) covered with a cover slip and sealed with rubbercement. The hybridization is carried out overnight at 37° C. in a humidchamber.

[0240] The detection of the hybrids is performed according to proceduresknown for in situ hybridizations on decondensed chromosomes (D. Pinkelet al., PNAS USA 83, 2934 (1986) and PNAS USA 85, 9138 (1988)).

[0241] Hybridized segments such as that shown in FIG. 10 are thenobserved by fluorescence microscopy. This example demonstrates thepossibility of detecting the presence of a gene on a DNA molecule, whichcan be used for diagnostic purposes or for physical mapping of thegenome.

1. Process for aligning a macromolecule (macromolecules) on the surfaceS of a support, characterized in that the triple line S/A/B (meniscus)resulting from the contact between a solvent A and the surface S and amedium B is caused to move on the said surface S, the saidmacromolecules having a part, especially an end, anchored on the surfaceS, the other part, especially the other end, being in solution in thesolvent A.
 2. Process according to claim 1, characterized in that themovement of the meniscus is achieved by evaporation of the solvent A. 3.Process according to claim 1, characterized in that the movement of themeniscus is achieved by relative movement of the A/B interface relativeto the surface S.
 4. Process according to claim 3, characterized in thatin order to move the meniscus, the surface S is removed from the solventA or the solvent A is removed from the surface S
 5. Process according toone of claims 1 to 4, characterized in that the meniscus is a water/airmeniscus.
 6. Process according to one of claims 1 to 5, characterized inthat the support consists, at least at the surface, of an organic orinorganic polymer, a metal, a metal oxide or sulfide, a semiconductorelement such as silicon or an oxide of a semiconductor element, or oneof the combinations thereof.
 7. Process according to one of claims 1 to6, characterized in that the support consists, at least at the surface,of glass, surface-oxidized silicon, gold, graphite, molybdenum sulfideor mica.
 8. Process according to one of claims 1 to 7, characterized inthat the support is in the form of a plate, bead, fiber or particlesystem.
 9. Process according to one of claims 1 to 8, characterized inthat the solvent A in which the macromolecules to be aligned are insolution is placed between two supports, of which one at leastcorresponds to the said support of surface S, and the meniscus is movedby evaporation.
 10. Process according to one of claims 1 to 9,characterized in that the anchoring of the macromolecule is carried outby physicochemical interaction, especially by adsorption or by covalentlinkage, either directly between the surface and the macromolecule, orindirectly between the surface and another molecule recognizing and/orinteracting with the said macromolecule.
 11. Process according to one ofclaims 1 to 10, characterized in that a support is used which has at thesurface an exposed reactive group having an affinity for the saidmacromolecule or a molecule with biological activity capable ofrecognizing the said macromolecule.
 12. Process according to one ofclaims 1 to 11, characterized in that the surface is coated with a groupchosen from the vinyl, amine, carboxyl, aldehyde or hydroxyl groups. 13.Process according to either of claims 10 and 12, characterized in thatthe surface contains: on a support, a substantially monomolecular layerof an organic compound of elongated structure having at least: anattachment group having an affinity for the support, and an exposedgroup having no or little affinity for the said support and the saidattachment group under attachment conditions, but optionally having,after chemical modification following the attachment, an affinity forthe said macromolecule or molecule with biological activity.
 14. Processaccording to one of claims 1 to 13, characterized in that the anchoringof a part of a macromolecule is carried out by adsorption on a surface,by bringing the said macromolecule into contact with the said surface ina determined region of pH or ionic content of the medium or by applyinga determined electric voltage on the anchoring surface.
 15. Processaccording to claims 1 to 14, characterized in that the pH for carryingout the anchoring is chosen in a range between a pH favoring a state ofcomplete adsorption and a pH favoring absence of adsorption.
 16. Processaccording to one of claims 1 to 15, in which the anchoring of a nucleicacid or of a protein is carried out by adsorption on a surface havinggroups containing ethylenic double bonds or amine groups, by bringingthe nucleic acid or the protein into contact with the surface in adetermined region of pH or ionic content of the medium.
 17. Processaccording to claim 16, characterized in that the anchoring ofnonfunctionalized DNA is carried out by adsorption on surfaces coatedwith molecules ending with a vinyl or amine group.
 18. Process accordingto claim 17, in which the anchoring of the DNA is carried out by its endonto a surface having groups with ethylenic double bonds, by bringingthe DNA into contact with the surface at a pH of less than
 8. 19.Process according to claim 18, characterized in that the reaction iscarried out at a pH of between 5 and 6, and is then stopped at pH
 8. 20.Process according to claim 17, characterized in that the anchoring ofDNA is carried out by its end onto a surface coated with polylysine or asilane group ending with an amine group.
 21. Process according to claim17, characterized in that the anchoring of the DNA is carried out by itsend onto a surface coated with an amine group by bringing the DNA intocontact with the surface at a pH of between 8 and
 10. 22. Processaccording to claim 17, characterized in that the anchoring of DNA iscarried out by its end onto a glass surface treated beforehand in anacid bath, by bringing the DNA into contact with the said surface at apH of between 5 and
 8. 23. Surface with aligned macromolecule(s)obtained by the process according to one of claims 1 to
 22. 24. Processfor detecting, separating and/or assaying a macromolecule in a sample,characterized in that a process for alignment according to one of claims1 to 23 is used in which a molecule with biological activity capable ofrecognizing the said sample macromolecule becomes attached to thesurface S, and in that the detection, separation or assay are carriedout using a reagent, fluorescent or otherwise, which detects thepresence of the attached molecule or the said macromolecule.
 25. Processaccording to one of claims 1 to 24, characterized in that the saidmacromolecule and molecule with biological activity are chosen fromproteins, nucleic acids, lipids, polysaccharides and derivativesthereof.
 26. Process according to claim 24, characterized in that thesaid macromolecule and molecule with biological activity are chosen fromantibodies, antigens, DNAs, RNAs, ligands or their receptors as well asderivatives thereof.
 27. Process according to claims 24 to 26,characterized in that the attached DNA contains the sequencecomplementary to a DNA sequence to be isolated from a sample. 28.Process according to claims 24 to 26, characterized in that the attachedprotein is capable of specifically recognizing and attaching a proteinto be isolated from a sample.
 29. Process according to claims 24 to 26,characterized in that the said molecule with biological activity ischosen from biotin, avidin, streptavidin, derivatives thereof or anantigen-antibody system.
 30. Process according to claims 24 to 26,characterized in that the surface exhibits low fluorescence and in thatthe reagent is fluorescent.
 31. Process according to claims 24 to 26,characterized in that the reagent consists of beads.
 32. Processaccording to one of claims 24 to 26, characterized in that the detectionis performed by optical or near field microscopy.
 33. Process accordingto one of claims 24 to 26, characterized in that the product of areaction between the molecule with biological activity and themacromolecule of the sample is subjected to stress in order to destroythe mismatches before the detection.
 34. Process for detecting amacromolecule consisting of a DNA sequence or a protein in a sample,according to one of claims 24 to 33, characterized in that: the samplecorresponding to solvent A, in which the said macromolecule is insolution, is brought into contact with the surface of the support underconditions for forming a DNA/DNA, DNA/RNA hybrid or for forming theprotein/protein reaction product, the hybrid or a macromolecule forlabeling the hybrid or the reaction product being anchored in one part,the remainder being in solution, it is stretched by the movement of themeniscus created by the contact between the solvent and the surface inorder to orient the hybrids or the said labeling macromolecules and themeasurement or the observation of the hybrids or of the said labelingmacromolecules thus orientated is carried out.
 35. Process according toone of claims 24 to 34, characterized in that the attached DNA and thesample DNA are “colored” differently and, after stretching, the positionof the complementary sequence relative to the end of the sample DNA ismeasured.
 36. Process according to one of claims 24 to 35, characterizedin that an ELISA or FISH detection method is used.
 37. Process accordingto one of claims 24 to 36, characterized in that the sample is theproduct or the substrate of an enzymatic amplification of nucleic acid.38. Process according to one of claims 24 to 37, characterized in thatthe DNA is stretched, then denatured and hybridized with specific probesin order to determine the position or the size of one or more determinedDNA sequences.
 39. Process for the physical mapping of a gene on agenomic DNA in which the DNA is aligned and/or detected according to aprocess of one of Clains 1 to
 38. 40. Process according to claim 39,characterized in that the position and the size of the desired gene onthe genomic DNA are determined by hybridization with probes specific forthe said gene to be mapped.
 41. Kit useful for carrying out a processaccording to either of claims 39 and 40 comprising: total genomic DNAfrom a reference host, a support having a surface permitting anchoringand alignnent of the DNA, probes specific for the gene to be mapped, andreagents for hybridization and detection of the DNA.
 42. Processaccording to one of claims 34 to 37, characterized in that the DNA isstretched, then denatured and hybridized with specific probes in orderto determine the presence or the absence of one or more given DNAsequences.
 43. Process for diagnosing a pathology related to thepresence or to the absence of a given DNA sequence specific for the saidpathology, in which a process according to claim 42 is used.
 44. Kituseful for carrying out a diagnostic process according to claim 43,characterized in that it contains: a support whose surface permits theanchoring and alignment of the patient's DNA, probes specific for thegene involved in the sought for pathology, and reagents forhybridization and detection of the DNA.
 45. Kit useful for carrying outa diagnostic process according to claim 43, characterized in that itcontains: a support whose surface has probes specific for the geneinvolved in the desired pathology, the said probes being anchored andaligned on the surface; reagents for labeling DNA, especially thepatient's DNA; reagents for the hybridization and the detection of theDNA.
 46. Process for preparing a gene from a genomic DNA, characterizedin that the position of the said gene on the genomic DNA aligned by theprocess of one of claims 1 to 27 is identified by means of a probespecific for the said gene and the enzymatic amplification of thesequence of the said gene and/or of its flanking sequences is carriedout and the amplified product is isolated.
 47. DNA construct containinga gene prepared by the process of claim 46, optionally associated with ahomologous or heterologous regulatory sequence.
 48. Use of a geneobtained by a process according to claim 46, for preparing a DNAconstruct useful in a process for replacing a gene in the genome of aneukaryotic cell by targeted insertion of a foreign gene, the saidforeign gene being obtained by the process of claim
 46. 49. Vectoruseful in a process for replacing a gene in the genome of a eukaryoticcell by targeted insertion of the said foreign gene, characterized inthat the said foreign gene is prepared according to the process of claim46.